PROJECT SUMMARY/ABSTRACT Staphylococcus aureus is a significant cause of morbidity and mortality due to the remarkable ability to colonize multiple host tissues. Consistent with this, S. aureus is the leading cause of skin and soft tissue infections, bacteremia, osteomyelitis and endocarditis. Treating these infections is challenging due to the prevalence of antibiotic resistant isolates, which necessitate the development of new therapeutic strategies. To proliferate within diverse tissues, S. aureus acquires essential nutrients by exploiting abundant nutrient reservoirs present in the host environment. S. aureus nutrient iron acquisition strategies have been studied for decades; however, the mechanisms employed by this pathogen to obtain the equally important nutrient sulfur during infection are not known. The sulfur-containing molecule, glutathione (GSH) is abundant in the host and supports in vitro proliferation of S. aureus. Whether GSH satisfies the sulfur requirement during pathogenesis is unresolved. This is because we do not know how S. aureus imports or catabolizes GSH or derivatives thereof. To begin to elucidate the mechanisms S. aureus uses to acquire host-derived GSH, we took a genetic approach and identified five transposon-disrupted mutants that failed to grow in medium supplemented with GSH as the sole source of sulfur. Notably, the transposons mapped to an operon that encodes a putative ABC transporter and GSH degradation enzyme. Consequently, we renamed the operon the GSH import system or gisABCD-ggt. These preliminary data represent identification of the first sulfur source acquisition system in S. aureus, and support the hypothesis that the GisABCD-Ggt is system is required for S. aureus utilization of host-derived GSH during infection. The proposed work will test this hypothesis by (i) defining the mechanisms that support S. aureus to import and catabolize host-derived GSH, and (ii) establishing the importance of Gis-mediated GSH acquisition to S. aureus pathogenesis. Understanding how infection and the host immune response affects the chemical nature of GSH is also a goal of this proposal. The completion of these studies will reveal the mechanisms S. aureus utilizes to acquire host-derived GSH during colonization of distinct tissues. This work has the potential to reveal a novel therapeutic strategy to combat antibiotic resistant S. aureus by impeding nutrient sulfur acquisition. We predict that the mechanism S. aureus employs to satisfy its sulfur requirement during infection is likely conserved in other bacterial pathogens, broadening the impact of the proposed work.